1. Field of the Invention
The present invention relates generally to the field of imaging. More particularly, it concerns techniques for enhancing contrast in optical techniques for biological applications.
2. Description of Related Art
Photonic technologies are playing an increasingly important role in biomedical diagnostics, resulting from improvements in optical instrumentation and an improved understanding of the interaction between light and biological tissues. At least two techniques have contributed to recent progress in biomedical optical imaging and disease diagnosis: (1) biological characterization techniques based on optical spectroscopy, and (2) imaging such as confocal microscopy and optical coherence tomographic (OCT), derived from interferometric and ultrafast optical technologies. In the first technique, highly specific in vivo identification of biological moieties at the molecular level may be accomplished using unique spectral signatures of intrinsic or extrinsic tissue chromophores. In the second, near-histological resolution imaging of sub-surface tissue microstructure may be performed even in highly scattering biological tissues by high-contrast rejection of multiply-scattered light.
Much work has been done to develop photonic technologies for diagnosing cancers and pre-cancers due to the fact that cancer is one of the leading causes of death in the United States and in the world. In the United States alone, deaths from cancer were estimated to number 560,000 in 1997. Currently, diagnosis and treatment of cancer follow histopathologic evaluation of directed biopsies. However, the tissue removal necessitated by these techniques not only may alter the progression of disease but is also very costly. Improving the capability for in-situ monitoring of disease progression may greatly enhance the ability to detect and treat cancer and pre-cancer.
In women, cervical cancer alone will account for an estimated 14,500 new cases and 4,800 deaths in 1997. Its precursor, cervical intraepithelial neoplasia (CIN), refers to a tumor growth in the cervical epithelium. Such growth is believed to originate from a single mutant cell near the basal layer of the epithelium. Rather than follow the normal maturation process, the mutant cell continues to divide without control. This results in a growing, undifferentiated group of cells, which gradually spreads from the basal layer to the surface layer of the epithelium. The disease is considered pre-invasive when it is confined to the epithelium. If detected at this stage, the disease is curable. Eventually, the basal layer may be compromised, and the transformed cells may invade the stroma. At this stage, the disease is considered invasive.
Early detection of CIN has been crucial to the reduction of the mortality caused by cervical cancer over the last decades. Currently, cytologic techniques are of widespread use in the screening of gynecologic cancers. Unfortunately, the accuracy of such techniques is not optimal. In one study of the accuracy of the Papanicolau test, it was found that sensitivity ranged from 11 to 99% and specificity from 14 to 97%. If an abnormal Pap smear is detected, further examination is often performed using a low power microscope called a colposcope. Colposcopy is used to identify the extent and grade of the lesion, and to direct any biopsies to be taken. While highly sensitive, colposcopy is unfortunately not very specific. This means that a large number of normal sites, usually inflammation or HPV infection, may be classified as diseased. Colposcopy is only performed by trained experts, making the cost of more widespread screening with this method prohibitive. In view of the above, there is a need for more accurate, automated screening and diagnostic tools to identify cervical precancers.
Work done in the area of optical spectroscopy has been aimed at aiding in the diagnosis of cancer and pre-cancer not only in gynecologic tissue but also in other kinds of tissue. Some techniques attempt to classify the tissue being probed by effectively assessing its biochemical and morphologic composition as evidenced by the light reflected, absorbed or emitted as Raman or fluorescence signals. Such techniques attempt to provide additional information that will result in improved in-situ diagnosis, allowing for better diagnostic performance by less-specialized personnel.
Confocal microscopy is an imaging technique well-suited for the evaluation of thick, turbid samples due to its ability to reject light from outside the focal volume. The technique has been used extensively in vitro and applications for in vivo and endoscopic use have been developed. Confocal microscopy has a spatial resolution on the order of 1 to 2 microns, allowing it to resolve single cells and their nuclei. This makes it a potential tool for aiding diagnosis since it may provide near-histologic resolution images in vivo. However, confocal imaging is only possible at depths of several hundred microns, as image quality is degraded by wavefront aberrations induced by the scattering in the tissue, and contrast may be reduced by multiply scattered, detected photons generated outside the confocal volume. There is therefore a need for techniques for enhancing contrast so as to improve confocal microscopy imaging.
Optical coherence tomography (OCT) is another imaging technique that may overcome at least some of the problems associated with multiple scattering, allowing it to image sub-surface structure in tissue. OCT detects very faint reflections of laser light directed into the tissue and determines at what depth these reflections occurred. This results in an image of the relative reflectivity of the tissue below the surface. This is related to the properties of individual cells as well as the overall structure of the tissue, both of which may change in the presence of disease.
With OCT, tomographic images of sub-surface biological microstructure may be obtained with xcx9c10 xcexcm spatial resolution. The heterodyne optical detection scheme inherent to OCT provides sensitivity to backscattered signals as small as one part in 1011 of the incident optical power; thus, extremely faint reflections may be visualized. In OCT, the specimen to be interrogated may be placed in the sample arm of an interferometer illuminated with a low-coherence light source. Interference between light returning from the reference arm and light scattered from internal sample reflections occurs only when the optical path lengths in both arms of the interferometer are matched to within the source coherence length. Thus, scanning the reference arm while monitoring the envelope of the interferometric signal generates a map of tissue reflectivity versus optical depth or xe2x80x9cA-scan,xe2x80x9d with axial resolution given by the coherence length. Cross-sectional images of tissue backscatter may be built from sequential A-scans obtained while scanning the probe beam across the tissue surface. Resulting two-dimensional data sets may be plotted as gray-scale images.
The initial clinical application of OCT was for high-resolution imaging of intraocular structure. OCT is well suited to ophthalmology because it is non-contact, easily adaptable to existing ophthalmic instrumentation, and the axial image resolution is independent of the working distance. A growing number of studies of OCT imaging in non-transparent media have also been reported. In vitro and in vivo studies have reported OCT imaging in the skin, teeth, and brain, as well as in vascular, respiratory, and gastrointestinal tissues. Recent technical advances in image acquisition time and probe miniaturization have produced the first studies on catheter and endoscopic OCT imaging in living animals and humans.
Using a catheter approximately 1 mm in diameter capable of insertion in an endoscope accessory channel, in vivo images of human esophagus, larynx, stomach, urinary bladder, and uterine cervix have been reported. Epithelial invasion of the basement membrane was distinctly visible in images of early cancers, implying that the technique may be promising for early diagnosis of tumors and precise guiding of excisional biopsy.
The success of OCT and other imaging techniques underscore many advantages in imaging subsurface morphology. However, OCT resolution is lower than that of light microscopy, the current gold standard in the tissue malignancy assessment following biopsy. Attaining the level of resolution necessary to make malignancy assessments in human epithelial tissue samples by nuclear size determination is currently unattainable through OCT. Therefore, if such assessments are to be done with OCT in-vivo, further differentiation techniques must be developed.
Despite the promise of imaging techniques discussed above, and their application to the detection of, for instance, cancer, little is understood about how to achieve optimal contrast in such images, particularly in highly-scattering tissues where early cancers and pre-cancers develop. The primary sources of signal in traditional OCT images arise from mismatches in tissue index of refraction; however, little is known about the wavelength-dependent microscopic fluctuations in the tissue refractive index and how those vary in normal and pathologic tissues.
In view of the above, it is apparent that a need exists for developing techniques to enhance contrast in different fields of biomedical imaging so that, for instance, cancers and pre-cancers may be diagnosed earlier and more easily, with greater accuracy.
In one respect, the invention is a method for enhancing contrast during imaging to assess cell and nuclear morphology of a sample. Between about 1% and about 10% by volume of acetic acid is applied to the sample. The sample is analyzed with an imaging device to create image data, the sample is diagnosed with the image data. As used herein, xe2x80x9cimage dataxe2x80x9d is to be read broadly to mean any data gathered by the imaging device. The use of the word xe2x80x9cimagexe2x80x9d is not meant to connote a graphical representationxe2x80x94rather, by xe2x80x9cimagexe2x80x9d and xe2x80x9cimagingxe2x80x9d, it is meant any information relating to the sample gathered by one or more devices. Image data may be a string of numbers, a graphical representation, or any other data known in the art.
In other respects, the method includes applying between about 3% and about 10% by volume of acetic acid to the sample. The imaging device may include a confocal microscope. The imaging device may include an optical coherence tomography apparatus. The imaging device may include a photon migration imaging device. The imaging device may include a two-photon excited fluorescence imaging device. The imaging device may include a spectroscopy apparatus. The spectroscopy may include reflectance spectroscopy. The spectroscopy may include absorption spectroscopy. The spectroscopy may include fluorescence spectroscopy. The spectroscopy may include Raman spectroscopy. The sample may be in vitro. The sample may be in vivo.
In another respect, the invention is a method for enhancing contrast during imaging to assess cell and nuclear morphology of a sample, wherein between about 0.5% and about 10% by volume of Toluidine blue is applied to the sample. The sample is analyzed with an imaging device to create image data, and the sample is diagnosed with the image data.
In another respect, the invention is a method for enhancing contrast during imaging to enhance edges of cells of a sample. Between about 2 to about 10 times physiological concentrations of hypertonic saline is applied to the sample. The sample is analyzed with an imaging device to create image data, and the sample is diagnosed with the image data.
In another respect, the invention is a method for enhancing contrast during imaging to enhance edges of cells of a sample, wherein between about 0.1 to about 0.5 times physiological concentrations of hypotonic saline is applied to the sample. The sample is analyzed with an imaging device to create image data, and the sample is diagnosed with the image data.
In another respect, the invention is a method for enhancing contrast during imaging to assess cell and nuclear morphology of a sample, wherein between about 5% and about 10% by volume of Lugol""s iodine is applied to the sample. The sample is analyzed with an imaging device to create image data, and the sample is diagnosed with the image data.
In another respect, the invention is a method for enhancing contrast during imaging to assess cell and nuclear morphology of a sample, wherein an absorbing dye is applied to the sample. The sample is analyzed with an imaging device to create image data, and the sample is diagnosed with the image data.
In other respects, the absorbing dye may include phycoerythrin. The absorbing dye may include indocyanine green. The absorbing dye may include lutetium texaphyrin. The analysis of the sample with an imaging device may include applying two or more illumination wavelengths to the sample, the scattering of the sample at the two illumination wavelengths being substantially different, analyzing the sample with an optical coherence tomography device to create image data, and diagnosing the sample with the image data.
In another respect, the invention is a method for enhancing contrast during imaging to assess cell and nuclear morphology of a sample, wherein a liposome is applied to the sample, the liposome containing a fluid of different refractive index. The sample is analyzed with an imaging device to create image data, and the sample is diagnosed with the image data.
In other respects, the fluid may include water. The fluid may include bovine serum albumin.
In another respect, the invention is a method for enhancing contrast during imaging to assess cell and nuclear morphology of a sample, wherein a contrast agent linked to a marker is applied to the sample. The sample is analyzed with an imaging device to create image data, and the sample is diagnosed with the image data.
In other respects, the marker may include CA125. The marker may include EGFR. The marker may include Her-2. The marker may include Annexin-V. The marker may include proliferating cellular nuclear antigen (PCNA). The marker may include endothelial growth factor receptor (EGFR). The marker may include vascular endothelial growth factor (VEGF). The marker may include human milkfat protein. The marker may include folate receptor.